Natural history of Ehrlichia ruminantium.

Ehrlichia ruminantium is an obligately intracellular proteobacterium which causes a disease known as heartwater or cowdriosis in some wild, and all domestic, ruminants. The organism is transmitted by ticks of the genus Amblyomma, and it is of serious economic importance wherever the natural vectors occur, an area which includes all of sub-Saharan Africa, and several islands in the Caribbean. The disease was first recognized in South Africa in the 19th century, where its tick-borne nature was determined in 1900, but the organism itself was not demonstrated until 1925, when it was recognized to be a rickettsia, initially named Rickettsia ruminantium. It was thus the first species of what are now known as Ehrlichia to be discovered, and most of the early work to elucidate the nature of the organisms, and its reservoirs and vectors, was performed in South Africa. The next milestone was the development, in 1945, of an infection and treatment regimen to immunize livestock, and this is still the only commercially available "vaccine" against the disease. Then in 1985, after fruitless attempts over many years, the organism was propagated reliably in tissue culture, opening the way for the first application of the newly developed techniques of molecular genetics. From 1990 onwards the pace of heartwater research accelerated rapidly, with notable advances in phylogeny, diagnosis, epidemiology, immunology, and vaccine development. The complete genome sequence was published in 2005, and during the last two years a new understanding has arisen of the remarkable genetic variability of the organism and new experimental vaccines have been developed. Despite all this the goal of producing an effective vaccine against the disease in the field still remains frustratingly just beyond reach. This article summarises our current understanding of the nature of E. ruminantium, at a time when the prospects for the development of an effective vaccine against the organism seem better than at any time since its discovery 83 years ago.

Identification of novel Babesia and Theileria species in South African giraffe (Giraffa camelopardalis, Linnaeus, 1758) and roan antelope (Hippotragus equinus, Desmarest 1804).

Blood specimens were received from five cases in which young adult giraffe, from different geographic origins in South Africa, showed sudden onset of disease and subsequently died. Additional specimens from two translocated giraffe, as well as one specimen from a roan antelope, were also included in the study. Blood slides from some of these animals showed the presence of piroplasms. DNA was extracted; the V4 hypervariable region of the 18S rRNA gene amplified and analyzed using the Reverse Line Blot (RLB) hybridization assay. PCR products failed to hybridize with any of the Babesia or Theileria species-specific probes, and only hybridized with the Babesia/Theileria genus-specific probe suggesting the presence of a novel species or variant of a species. Full-length 18S rDNA was amplified, cloned and the recombinants were sequenced. 18S rRNA gene sequence similarity analysis revealed the presence of novel piroplasm species in both healthy giraffe and a roan antelope and clinically sick or dead giraffe. Phylogenetic analysis grouped five of these organisms in the Babesia sensu stricto clade and three in the Theileria sensu stricto clade. Although parasites were observed in blood smears, there is no direct evidence that piroplasmosis caused the death of five giraffe, although it certainly seems to be likely.

Molecular sequence evidence for the reclassification of some Babesia species.

Taxonomic characterization of organisms in the genera Theileria and Babesia was originally based on observations of morphology and certain general phenotypic characteristics, which enabled many parasites to be unequivocally assigned to a particular genus. However, application of molecular genetic techniques, such as the polymerase chain reaction (PCR) for gene amplification, and DNA sequencing, have revealed gross inconsistencies in the assignation of some parasite genetic variants, particularly those of the B. gibsoni and B. microti complexes, to the genus Babesia. These variants cannot be assigned, on the basis of sequence information and phylogenetic analysis, to either of the genera Theileria and Babesia. The gene for which most sequence information is available for phylogenetic analysis is the small subunit ribosomal RNA (srRNA) gene. This gene allows clear distinction of the genera Theileria and Babesia (sensu stricto) and reveals that many "Babesia" variants are phylogenetically distinct from both genera. This distinction is confirmed, for some of the variants, by beta-tubulin sequence data, suggesting that the organisms should be renamed and reclassified.

An attenuated Ehrlichia ruminantium (Welgevonden stock) vaccine protects small ruminants against virulent heartwater challenge.

Heartwater is a tick-borne disease of ruminants caused by the intracellular rickettsia Ehrlichia ruminantium. The only commercially available immunization procedure involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by treatment with tetracyclines when fever develops. The virulent Welgevonden stock of E. ruminantium was attenuated by continuous propagation of the organisms in a canine macrophage-monocyte cell line (DH82), followed by re-adaptation to grow in a bovine endothelial cell line (BA 886). The material used for the present experiments consisted of the attenuated stock between passages 43 and 64 after re-adaptation. When inoculated into sheep or goats the attenuated organisms did not produce disease, and the only symptom observed was a rise in body temperature in most, but not all, animals. All sheep injected with 2 ml of culture suspension were subsequently found to be fully protected against a lethal needle challenge with the virulent homologous stock or with one of four different heterologous stocks (Ball 3, Gardel, Mara 87/7, Blaauwkrans). Titrations of elementary body suspensions showed that 2ml of a 1:10,000 dilution of culture suspension injected into sheep or goats was still sufficient to trigger an immune response which resisted a lethal needle challenge with the virulent Welgevonden stock. Adult Amblyomma hebraeum ticks, fed as nymphs on sheep immunized with DH82-derived organisms of passage 111, were able to transmit the attenuated stock to a naive sheep, which was found to be protected against a subsequent lethal homologous needle challenge.

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number.

Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.

Characterization of a major outer membrane protein multigene family in Ehrlichia ruminantium.

Ehrlichia ruminantium is a tick-transmitted rickettsial pathogen, which causes heartwater or cowdriosis in wild and domestic ruminants. A dominant antibody response of animals infected with E. ruminantium is directed against the outer membrane protein MAP1 (major antigenic protein 1). Part of the locus containing map1 has been characterized and consists of four map1 paralogs, designated map1-2, map1-1, map1 and map1+1, indicating that map1 is encoded by a multigene family. The purpose of this study was to determine the total number of map1 paralogs and their transcriptional activities. Using genome walking and data from an ongoing E. ruminantium genome sequencing project at the Onderstepoort Veterinary Institute, we found 16 paralogs of the map1 gene tandemly arranged in a 25 kb region of the E. ruminantium genome. The map1 multigene family is downstream of a hypothetical transcriptional regulator gene and upstream of the secA gene. Thirteen paralogs at the 5' end of the 25-kb locus were connected by short intergenic spaces (ranging from 0 to 42 bp) and the remaining three paralogs at the 3' end were connected by longer intergenic spaces (ranging from 375 to 1612 bp). All 16 map1 paralogs were transcriptionally active in E. ruminantium grown in endothelial cells and paralogs with short intergenic spaces were co-transcribed with their adjacent genes.

Development of improved vaccines for heartwater.

Heartwater is a tick-borne disease of ruminants which causes major economic losses for domestic livestock owners throughout sub-Saharan Africa and the Caribbean. It is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium and the only commercially available vaccination procedure is over 50 years old. It involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by antibiotic treatment when fever develops. Experimental attenuated, inactivated, and nucleic acid vaccine procedures have been investigated over the last half-century, but none of them has yet been particularly successful. We have developed two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuated vaccine was developed by continuous passage of E. ruminantium organisms of the virulent Welgevonden isolate in a continuous canine macrophage-monocyte cell line. After more than 50 passages the cultures produced no disease when inoculated into mice or sheep, and the inoculated animals were 100% immune to a subsequent lethal homologous needle challenge. The nucleic acid vaccine is based on four E. ruminantium genes from a genetic locus involved in nutrient transport. A cocktail of all four genes, cloned in a DNA vaccine vector and used to immunize sheep, engendered 100% protection against a subsequent lethal needle challenge with the homologous isolate and with each of five different virulent heterologous isolates. Sheep immunized with this cocktail were also exposed to a field challenge in a heartwater-endemic area and few animals survived. This suggests that the local E. ruminantium genotypes were different from any which were administered by needle challenge, or that needle challenge is not a good model for tick challenge in the field.

Preparation of Ehrlichia ruminantium challenge material for quantifiable and reproducible challenge in mice and sheep.

The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.

Molecular diagnosis of theileriosis and heartwater in bovines in Africa.

The advent of the polymerase chain reaction (PCR) coupled with the specificity of deoxyribocucleic acid (DNA)-DNA hybridization has led to the development of specific and sensitive molecular diagnostic tests to detect and characterize the organisms that cause theileriosis and heartwater. Theileriosis is a widespread disease of wild and domestic ruminants caused by apicomplexan parasites of the genus Theileria. Species-specific variations in small subunit ribosomal ribonucleic acid genes (SSUrRNA) have been used to develop probes that can distinguish between Theileria species such as T. parva, T. annulata, T. mutans, T. buffeli and T. taurotragi. Routine application of this test has led to the discovery of previously unknown species, such as Theileria sp. (buffalo) which is apparently apathogenic to both buffalo and cattle, and Theileria sp. (sable) which is pathogenic to sable and possibly also to roan antelope. In addition, characterization probes located in the internal transcribed spacer (ITS) can be used to distinguish between most isolates of the causative agents of East Coast fever (T. p. parva) and Corridor disease (T. p. lawrencei). Heartwater is an economically important disease of livestock and some wild ruminants, caused by the intracellular rickettsial parasite Ehrlichia (ex Cowdria) ruminantium. DNA probes used to detect and characterize E. ruminantium isolates include SSUrRNA (16S) probes, the pCS20 probe and map1 probes. A panel of eight 16S probes has been developed for the detection of E. ruminantium and related Ehrlichia species. There are probes for 5 different E. ruminantium genotypes, one which will detect all 5 of these genotypes, one to detect any Ehrlichia species other than E. ruminantium, and one for any Anaplasma species. The pCS20 probe is specific for E. ruminantium and is the most sensitive of the probes for E. ruminantium detection, but it is not able to distinguish among the different genotypes. The map1 gene has also been used for diagnosis, but the extensive polymorphism of this gene means that it is most useful for characterization of different genotypes of the parasite. Routine application of these tests has led to the discovery of new genotypes that are probably not E. ruminantium but are probably new species of Ehrlichia.